Acetic Acid Enables Precise Tailoring of the Mechanical Behavior of Protein-Based Hydrogels

Engineering viscoelastic and biocompatible materials with tailored mechanical and microstructure properties capable of mimicking the biological stiffness (<17 kPa) or serving as bioimplants will bring protein-based hydrogels to the forefront in the biomaterials field. Here, we introduce a method that uses different concentrations of acetic acid (AA) to control the covalent tyrosine–tyrosine cross-linking interactions at the nanoscale level during protein-based hydrogel synthesis and manipulates their mechanical and microstructure properties without affecting protein concentration and (un)folding nanomechanics. We demonstrated this approach by adding AA as a precursor to the preparation buffer of a photoactivated protein-based hydrogel mixture. This strategy allowed us to synthesize hydrogels made from bovine serum albumin (BSA) and eight repeats protein L structure, with a fine-tailored wide range of stiffness (2–35 kPa). Together with protein engineering technologies, this method will open new routes in developing and investigating tunable protein-based hydrogels and extend their application toward new horizons.

and re-suspended in elution/ washing (E/W) buffer (NaH 2 PO 4 50 mM, NaCl 300 mM, DTT 1mM, glycerol 5% v/v, pH 7.0) and lysed with lysozyme, DNase, and RNase in the presence of protease inhibitors, followed by sonication. After cell lysis, the soluble protein fraction was passed through a chemical affinity purification NiNTA column, then eluted with E/W buffer containing 250 mM Imidazole. We used size exclusion chromatography to extract the exact pL-8 structure (Akta GE, elution in HEPES 50mM, NaCl 150 mM, pH 7.2 buffer). Then, we concentrated the pL-8 to the final concentration of 2 mM using protein concentrator columns.
Protein-based hydrogel synthesis. Phosphate buffer solutions containing NaH 2 PO 4 ~ 10mM, NaCl ~ 150 mM, with different AA concentrations (0.25 -1.5 % (v/v)) were first prepared and then balanced to pH ~ 7.4 using KOH (1 M) solution. Then, the bovine serum albumin (BSA) or p-L8 protein were dissolved in these solutions at 2-and 1-mM concentrations, respectively. The protein solution, APS, and [Ru(bpy) 3 ] 2+ were mixed at a volume ratio of 15:1:1. Subsequently, the mixture was centrifuged to remove air bubbles and then loaded into a transparent Teflon tube, as reported previously. [10,35] The Teflon tube containing the hydrogel mixture was placed under white light for 30 minutes at room temperature (RT). Protein-based hydrogel samples without AA were synthesized as reported in our previous study. [10,35] Afterward, the hydrogel sample was removed from the tube and immersed in TRIS solution (20 mM Tris, 150 mM NaCl, pH~ 7.4) to equilibrate.
ATR-FTIR Measurements. FTIR spectra of BSA solution and hydrogel samples were recorded using Nicolet iS50 FT-IR in attenuated total reflection (ATR) mode equipped with a round Diamond, Type IIa crystal. 16 scans were recorded for each sample measurement with a nominal resolution of 8 cm -1 . The content of three main secondary structures of BSA, including intramolecular β-sheets (1610-1630 cm -1 ), -helix (1648-1660 cm -1 ), and β-turns (1660-1689 cm -1 ), was analyzed by the spectral deconvolution of the Amide I band (1600 to 1700 cm -1 ), according to previous studies. 42,44,54,55 Quantifying the effect of AA on Tyrosine-Tyrosine crosslinks within BSA-hydrogel samples. Triplicates of hydrogel samples with different AA concentrations (0%, 0.5%, 0.75%, 1%, and 1.5% (v/v)) were prepared as described previously. Each hydrogel sample was placed in a sealed Eppendorf tube of 1 mL HCl (6 N) and kept at 105 °C for 2 h, using a heat block to ensure full hydrolysis of amide bonds. Following treatment, 100 μL of acid hydrolysis product from each sample was transferred to a new 1.5 mL Eppendorf tube and neutralized by NaOH (5 M). After that, 0.1 M Na 2 CO 3 −NaHCO 3 buffer (pH ~ 9.9) was added to the neutralized solutions up to 1 mL final volume. The fluorescence emission intensity was recorded at 410 nm using the infinite M200 spectrophotometer (TECAN) plate-reader.
Mechanical Characterization. The mechanical characterization of the BSA and L8 -based hydrogel samples was performed by a force-clamp rheometer machine, as reported in our previous studies. Hydrogel samples were subjected to a force-ramp protocol with a controlled stress/relaxation rate of 0.01 mN/s (40 Pa/s) at room temperature while immersed in TRIS or 6 M GuHCl solution. Young's modulus was calculated from the slope over the elastic region of each stress-strain curve. The energy dissipation was calculated from the hysteresis area enclosed by the stress-strain curves.
Swelling ratio measurements. For water content experiments, triplicates of BSA-based hydrogels containing different concentrations of AA (0 -1.5% (v/v)) were synthesized as described in the previous section and then soaked in TRIS buffer (20 mM and 150 mM NaCl, pH~7.4) at 4°C for 24 h. After that, the hydrogels were removed from the TRIS solution, blotted with filter paper to remove excess buffer, and then weighed to obtain each sample's wet weight ( ). Then, the same hydrogels were desiccated in a vacuum chamber for 24 h before weighing to obtain each sample's dry weight ( ). The swelling ratio was calculated using the following equation .